Cryptococcus neoformans is a ubiquitous environmental fungus that is a major cause of disease in immunocompromised patients. Following inhalation, there is propensity for yeast cells to invade the central nervous system, which can result in meningoencephalitis. A central question in the pathogenesis of this fungus is how it adapts to and proliferates in the harsh environment of the human host. Three major virulence factors have been identified: an anti-phagocytic polysaccharide capsule, antioxidant melanin pigments, and the complex trait of thermotolerance. Preliminary results suggest the existence of an additional virulence factor: increased genetic instability at the human host temperature, which can contribute to rapid adaptation. Temperature-dependent instability has been observed with cultured yeast cells and, at the two reporter genes examined to date, is due to insertional inactivation by transposable elements (TEs). C. neoformans is estimated to contain ~30 distinct TEs, but these elements have not been well characterized. The goal of the proposed experiments is to define the extent/nature of temperature-dependent TE mobilization in free-living yeast cells, and to examine whether similar mobilization occurs in the context of model host systems. Thus far, movement of both a DNA transposon and a retrotransposon has been detected using a single selective agent. Aim 1 will use other selective systems to probe for the mobilization of additional TEs. As the first step in characterizing TE movement on a global scale, Aim 2 will use the long reads produced by PacBio sequencing to annotate all elements in the genome. In Aim 3, transposon-accumulation lines obtained by sub-culturing yeast cells will be subjected to whole genome sequencing in order to define the extent of TE movement globally. Finally, Aim 4 will employ three host models (the greater wax moth Galleria mellonella, cultured murine macrophages, and nasally-infected mice) to document similar TE mobilization in the context of complex fungal-host interactions. Results obtained from these analyses will provide basic insight into how C. neoformans rapidly adapts to the human host to cause disease, which may in turn inform the development of novel therapeutic interventions.